Characterizing the interaction between pyrogallol and human serum albumin by spectroscopic and molecular docking methods

In the present study, the interaction of Pyrogallol (PG) with human serum albumin (HSA) was investigated by UV, fluorescence, Circular dichroism (CD), and molecular docking methods. The results of fluorescence experiments showed that the quenching of intrinsic fluorescence of HSA by PG was due to a static quenching. The calculated binding constants (K) for PG-HSA at different temperatures were in the order of 10⁴?M ^?1, and the corresponding numbers of binding sites, n were approximately equal to unity. The thermodynamic parameters, ΔH and ΔS were calculated to be negative, which indicated that the interaction of PG with HSA was driven mainly by van der Waals forces and hydrogen bonds. The negative value was obtained for ΔG showed that the reaction was spontaneous. In addition, the effect of PG on the secondary structure of HSA was analyzed by performing UV–vis, synchronous fluorescence, and CD experiments. The results indicated that PG induced conformational changes in the structure of HSA. According to Förster no-radiation energy transfer theory, the binding distance of HSA to PG was calculated to be 1.93?nm. The results of molecular docking calculations clarified the binding mode and the binding sites which were in good agreement with the results of experiments.

Communicated by Ramaswamy H. Sarma     

On CK2 regulation of chronic lymphocytic leukemia cell viability

Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, β2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.     

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.     

Analysis of the molecular variation between and within cultivated and wild Pistacia species using AFLPs

Knowledge of pistachio genetic diversity is necessary for the formulation of appropriate management strategies for the conservation of these species. We analysed amplified fragment length polymorphisms in a total of 216 pistachio accessions, which included seven populations from three wild species (Pistacia vera, Pistacia khinjuk and Pistacia atlantica subsp. kurdica) and most of the important cultivars from Iran, together with some foreign cultivars. High levels of genetic diversity were detected within the Iranian cultivars, and they showed a clear separation from foreign cultivars, as revealed by unweighted pair group method with arithmetic averaging and supported by analysis of molecular variance. The lowest amount of polymorphism was observed in P. atlantica subsp. kurdica, which showed the lowest number of total bands as compared to the other species. This revealed strong genetic erosion of P. atlantica subsp. kurdica, which reflected a severe decline in habitat and over-exploitation. Based on these findings, strategies are proposed for the genetic conservation and management of pistachio species and cultivars.     

How Modification of Accessible Lysines to Phenylalanine Modulates the Structural and Functional Properties of Horseradish Peroxidase: A Simulation Study

Horseradish Peroxidase (HRP) is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241). In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca²⁺ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174) and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F′F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42) and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved concurrent with a decrease in enzyme activity, upon manipulation of charge/hydrophobicity balance at the protein surface.     

Comparison of the effects of HGF,BDNF, CT‐1, CNTF,and the branchial arches on the growth of embryonic cranial motor neurons

In the developing embryo, axon growth and guidance depend on cues that include diffusible molecules. We have shown previously that the branchial arches and hepatocyte growth factor (HGF) are growth‐promoting and chemoattractant for young embryonic cranial motor axons. HGF is produced in the branchial arches of the embryo, but a number of lines of evidence suggest that HGF is unlikely to be the only factor involved in the growth and guidance of these axons. Here we investigate whether other neurotrophic factors could be involved in the growth of young cranial motor neurons in explant cultures. We find that brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and cardiotrophin‐1 (CT‐1) all promote the outgrowth of embryonic cranial motor neurons, while glial cell line‐derived neurotrophic factor (GDNF) and neurotrophin‐3 (NT‐3) fail to affect outgrowth. We next examined whether HGF and the branchial arches had similar effects on motor neuron subpopulations at different axial levels. Our results show that HGF acts as a generalized rather than a specific neurotrophic factor and guidance cue for cranial motor neurons. Although the branchial arches also had general growth‐promoting effects on all motor neuron subpopulations, they chemoattracted different axial levels differentially, with motor neurons from the caudal hindbrain showing the most striking response. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 101–114, 2002     

Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: ionic channels and gamma-glutamyl transpeptidase activity

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.     

Preparation of superparamagnetic iron oxide/doxorubicin loaded chitosan nanoparticles as a promising glioblastoma theranostic tool

Theranostic nanoparticles (NPs) are promising for opening new windows toward personalized disease management. Using a single particle capable of both diagnosis and drug delivery, is the major benefit of such particles. In the present study, chitosan NPs were used as a dual action carrier for doxorubicin (DOX; chemotherapeutic agent) and superparamagnetic iron oxide nanoparticles (SPIONs; imaging agent). SPIONs and DOX were loaded at different concentrations within poly-l -arginine-chitosan-triphosphate matrix (ACSD) using the ionic gelation method. NPs’ size were in the range of 184.33 ± 4.4 nm. Drug release analysis of DOX loaded NPs (NP-DOX) showed burst release at pH 5.5 (as in tumor environment) and slow release at pH 7.4 (physiological condition), demonstrating pH-sensitive drug release profile. NP-DOX internalization was confirmed by flowcytometry and fluorescent microscopy. Uptake process results were corroborated by accumulation of drug in the intracellular space. Iron content was evaluated by inductively coupled plasma and prussian blue staining. In vitro magnetic resonance imaging (MRI) showed a decline in T ₂ relaxation times by increasing iron concentration. MRI analysis also confirmed uptake of NPs at the optimum concentration in C6 glioma cells. In conclusion, ACSD NPs could be utilized as a promising theranostic formulation for both diagnosis and treatment of glioblastoma.     

Acellular bone marrow extracts significantly enhance engraftment levels of human hematopoietic stem cells in mouse xeno-transplantation models

Hematopoietic stem cells (HSC) derived from cord blood (CB), bone marrow (BM), or mobilized peripheral blood (PBSC) can differentiate into multiple lineages such as lymphoid, myeloid, erythroid cells and platelets. The local microenvironment is critical to the differentiation of HSCs and to the preservation of their phenotype in vivo. This microenvironment comprises a physical support supplied by the organ matrix as well as tissue specific cytokines, chemokines and growth factors. We investigated the effects of acellular bovine bone marrow extracts (BME) on HSC in vitro and in vivo. We observed a significant increase in the number of myeloid and erythroid colonies in CB mononuclear cells (MNC) or CB CD34+ cells cultured in methylcellulose media supplemented with BME. Similarly, in xeno-transplantation experiments, pretreatment with BME during ex-vivo culture of HSCs induced a significant increase in HSC engraftment in vivo. Indeed, we observed both an increase in the number of differentiated myeloid, lymphoid and erythroid cells and an acceleration of engraftment. These results were obtained using CB MNCs, BM MNCs or CD34(+) cells, transplanted in immuno-compromised mice (NOD/SCID or NSG). These findings establish the basis for exploring the use of BME in the expansion of CB HSC prior to HSC Transplantation. This study stresses the importance of the mechanical structure and soluble mediators present in the surrounding niche for the proper activity and differentiation of stem cells.